Abstract
Antigen Used for Immunization
Purified, classical swine fever virus (Paderborn strain) recombinant E2 protein produced in baculovirus.
Method of Immunization
The purified recombinant CSFV E2-Paderborn protein was used as the immunogen for 4- to 6-week-old female BALB/c mice. 1 Specifically, 20 μg of the protein were mixed with the adjuvant Quillaja saponaria A and injected at 4-week intervals into mice intraperitoneally for the primary injection and subcutaneously for two additional immunizations. A total of 5 μg of unadjuvanted CSFV E2-Paderborn was administered intravenously and intraperitoneally 3 days prior to euthanizing the mice.
Parental Cell Line Used for Fusion
P3X63-Ag8.653 myeloma cell line.
Selection and Cloning Procedure
Hybridomas were selected using semi-solid HAT agar. Positive hybridomas were subcloned by end-point dilution.
Heavy and Light Chains of Immunoglobulin
IgG 1/k, IgG 2a/k, IgG 2b/k.
Specificity
Various reactivity patterns were identified using immunoperoxidase staining of cells infected with different classical swine fever virus strains. In addition to BVDV and BDV, there was no reactivity observed with the Alfort strain with any of the G series MAbs. Monoclonal antibody 5-2 did not react with any of the virus-infected cells at any concentration whereas MAb 25-2 only reacted with Paderborn-infected cells at the highest antibody concentration (1,290 μg IgG/mL) tested. Similarly, MAbs 35-2 and 39-1 also only showed reactivity with Paderborn-infected cells at the highest IgG concentration tested (i.e., 2000 μg/mL and 1400 μg/mL, respectively); however, their specificity could not be fully determined since testing was not performed with all the strain used in this study. Monoclonal antibody 31-1 demonstrated the next broadest reactivity pattern, which included cells infected with all strains, except Alfort, followed by G series MAbs 34-1, which reacted with all cells infected with all strains, except Alfort and Kanagawa, and 41-1, which reacted with cells infected with all strains, except Alfort and Peru (which reacted with cells infected with all strains, except Paderborn and Parma). Several MAbs (11-1, 28-2, 37-3, 38-2) reacted with strains representing genetic groups 1.3 (Honduras), 2.1 (Paderborn), 2.2 (Parma), 2.3 (Diepholtz), and 3.1 (congenital tremor) but did not react with other representative genetic group 1 (Alfort and Peru) or group 3 (Kanagawa) strains. It was interesting to note that MAb 14-1 reactivity was restricted to cells infected with representative strains (Paderborn, Parma, and Diepholtz) from genetic group 2, although only at the highest IgG concentration (592 μg/mL) tested for Diepholtz-infected cells.
The specificity of these MAbs was also determined by indirect ELISA. The CSFV Alfort or Paderborn-specific ELISA was performed by immobilizing either E. coli-expressed truncated recombinant Alfort/187-E2 (recAlfort E2) or baculovirus-expressed Paderborn-E2 (recPaderborn E2) antigens on microtiter plates as previously described. The antigen immobilized on a microtiter plate provided in the Herdcheck CSFV Ab test kit (IDEXX Europe, Koolhovenlaan, Netherlands) was prepared as per instructions with some slight modifications. Detection of MAb reactivity was achieved by using the chromogenic substrate provided with the kit.
All of the MAbs reacted with the antigen in the IDEXX assay, although MAb 25-2 demonstrated reactivity at only the highest IgG concentration (1290 μg/mL) tested. All the MAbs reacted with the baculovirus virus-expressed Paderborn E2 protein (recPaderborn E2). Only four of the G series MAbs (5-2, 11-1, 31-1, 41-1) reacted with the E. coli-expressed truncated Alfort E2 protein (recAlfort E2).
Characterization of CSFV-E2 MAbs by Western blot reactivity indicated that all the G series MAbs reacted with the recPaderborn E2 protein, and for MAbs 14-1, 28-2, 34-1, 37-3, and 38-2, this was the only positive reaction recorded. Monoclonal antibodies 5-2 and 25-2 reacted with all three recombinant E2 proteins. In addition to reacting with the recPaderborn E2 protein, MAbs 11-1, 31-1, and 41-1 reacted with the recBrescia E2 protein and MAbs 35-2 and 39-1 reacted with the recKanagawa E2 protein.
Specific Antigen Identified
E2 protein of classical swine fever virus.